Power and limitations of environmental DNA metabarcoding for surveying leaf litter eukaryotic communities

Leaf litter habitats shelter a great variety of organisms, which play an important role in ecosystem dynamics. However, monitoring species in leaf litter is challenging, especially in highly diverse environments such as tropical forests, because individuals may easily camouflage themselves or hide in the litter layer. Identifying species based on environmental DNA (eDNA) would allow us to assess biodiversity in this microhabitat, without the need for direct observation of individuals. We applied eDNA metabarcoding to analyze large amounts of leaf litter (1 kg per sample) collected in the Brazilian Atlantic forest. We compared two DNA extraction methods, one total and one extracellular, and amplified a fragment of the mitochondrial 18S rRNA gene common to all eukaryotes, to assess the performance of eDNA from leaf litter samples in identifying different eukaryotic taxonomic groups. We also amplified two fragments of the mitochondrial 12S rRNA gene to specifically test the power of this a...